Mosaic organization and heterogeneity in frequency of allelic recombination of the Plasmodium vivax merozoite surface protein-1 locus

Abstract

The organization and allelic recombination of the merozoite surface protein-1 gene of Plasmodium vivax (PvMsp-1), the most widely prevalent human malaria parasite, were evaluated in complete nucleotide sequences of 40 isolates from various geographic areas. Alignment of 31 distinct alleles revealed the mosaic organization of PvMsp-1, consisting of seven interallele conserved blocks flanked by six variable blocks. The variable blocks showed extensive variation in repeats and nonrepeat unique sequences. Numerous recombination sites were distributed throughout PvMsp-1, in both conserved blocks and variable block unique sequences, and the distribution was not uniform. Heterozygosity of PvMsp-1 alleles was higher in Asia (0.953 +/- 0.009) than in Brazil (0.813 +/- 0.047). No identical alleles were shared between Asia and Brazil, whereas all but one variable block nonrepeat sequence found in Brazil occurred in Asia. These observations suggest that P. vivax populations in Asia are ancestral to Brazilian populations, and that PvMsp-1 has heterogeneity in frequency of allelic recombination events. Recurrent origins of new PvMsp-1 alleles by repeated recombination events were supported by a rapid decline in linkage disequilibrium between pairs of synonymous sites with increasing nucleotide distance, with little linkage disequilibrium at a distance of over 3 kb in a P. vivax population from Thailand, evidence for an effectively high recombination rate of the parasite. Meanwhile, highly reduced nucleotide diversity was noted in a region encoding the 19-kDa C-terminal epidermal growth factor-like domain of merozoite surface protein-1, a vaccine candidate.

Fig 1.

Sequence variation in PvMsp-1 alleles. Amino acid sequences of 40 isolates and two monkey-adapted strains (Sal-1 and Belem strains) of P. vivax are aligned. Dots and dashes represent residues identical to Sal-1 and deletions, respectively. Asterisks denote residues identical to either the Sal-1 or Belem sequence. Other types of substitutions are listed under “Others.” Interallele variable blocks are boxed; repeats and polymorphic residues are indicated by <, >, and X, respectively; and boundaries for subblocks are demarcated by dotted vertical lines. Codons with synonymous nucleotide substitutions are indicated by lowercase letters. Bold letters with and without underlining denote residues conserved perfectly among P. vivax, P. falciparum (K1 allele and MAD20 allele), P. chabaudi, P. berghei, and P. yoelii, and residues conserved among P. vivax and three of the other four species, respectively. Dotted underlining indicates the sites of Rm (22).

Fig 2.

Allelic variation in PvMsp-1 represented by associations of sequences of six variable blocks. Variable blocks 2, 6, 8, and 10 contain short tandem repeats: block 2, degenerating 5-mer (GSXXX)_n=1–9 (X stands for any residue); block 6, poly (Q)_n=10–21; block 8, degenerating repeats of (PVTTTX)_n=2 or (PXVAPXX)_n=2 (italicized); block 10, degenerating 5-mer (VPXXX)_n=5–8. Variable blocks 4 and 12 and subblocks flanking these repeats have unique sequences, with each indicated by filled, unfilled, diagonal bricked, or checkerboard patterns. To simplify presentation, patterns are used to indicate different sequences within each block; i.e., a given pattern does not represent the same sequence in different blocks. In block 12, a small conserved region is excluded. Assignment of 42 isolates into 31 allelic types is as follows: 1 = Sal-1; 2 = BP29; 3 = BP1; 4 = BP13, BP63; 5 = BP30; 6 = BR07; 7 = BP39, BR44; 8 = T064, TF14, TF127; 9 = TV400; 10 = TG57; 11 = TC22; 12 = TG44; 13 = T107; 14 = T124; 15 = TFF18; 16 = T131, TG40; 17 = TC28, TC103; 18 = TG55; 19 = TD29; 20 = TG46; 21 = T077, TG48; 22 = TE26; 23 = BD4; 24 = BD1, BD2, BD9; 25 = BD6; 26 = IN1; 27 = SK1, SK2, SK4; 28 = SK3; 29 = VM55; 30 = VM278; 31 = Belem. Complete nucleotide identity occurs within each association type, except type 8, in which three isolates show substitutions at codons 1,564 and 1,705 (Fig. 1).

Fig 3.

Proportions of linkage disequilibrium between polymorphic sites within PvMsp-1 in a population from Thailand. Percentages of synonymous sites (2,346 pairs, stippled bar) and nonsynonymous sites (5,265 pairs, filled bar) that had statistically significant linkage disequilibrium (P < 0.05) are shown according to the nucleotide distance between the sites.

Fig 4.

Alignment of block 12 of PvMsp-1 alleles and Msp-1 of P. cynomolgi and P. knowlesi. P. vivax basic types 1–4 form mosaic mixtures of recombinant types 5–10. Note that P. cynomolgi sequence mostly consists of the four basic types of P. vivax. Residues conserved among P. vivax alleles are boxed, and those unique to P. cynomolgi and/or P. knowlesi are shaded. Symbols are: b = E or K, x = D or A, and z = K or T.